Neuroprotective composition and uses thereof

ABSTRACT

A neuroprotective composition for protecting neuronal cells against oxidative stress and methods for using and preparing the same. More particularly, the neuroprotective composition of the invention comprises a mixture of pyruvate, antioxidant, and lipid(s) such as fatty acids. The neuroprotective composition could be used for the treatment of brain trauma, brain or cerebrovascular ischemia, neurodegenerative diseases, poisoning of neuronal cells, the diminution of drugs side effects and for preservation of neuronal grafts.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the use of an amphiphilicantioxidant composition as a neuroprotective agent and to methods forusing and preparing the same. More particularly, the present inventionpertains to the use of a formulation of pyruvate, antioxidant, andlipid(s) such as fatty acids for protecting neurons against oxidativestress.

[0003] 2. Description of the Prior Art

[0004] Reactive oxygen species (ROS) have been implicated in thedevelopment of many heart and brain dysfunctions. Ischemia/reperfusioninsults to these organs are among the leading causes of mortality inAmerica. These insults are caused by complete or partial localocclusions of heart and brain vasculature, by heart stroke or attack,and by cerebral attacks and trauma to the brain. In addition, ROS areinvolved in artherosclerotic lesions, in the evolution of variousneurodegenerative diseases, and are also produced in association toepileptic episodes, in inflammation, in the mechanisms of action ofvarious neurotoxicants, or as side-effects of drugs.

[0005] Until now, no ideal therapeutic agent is known to protectneuronal cells against oxidant species associated with various types ofoxidative stress. It would therefore be highly desirable to have suchneuroprotective agent.

[0006] TRIAD is combination of pyruvate, antioxidant and fatty acids.This composition has been patented in 1997 in the U.S. as a therapeuticwound healing compositions (U.S. Pat. No. 5,652,274). Many related U.S.patents have also been issued for covering the uses of TRIAD inantikeratolytic compositions (U.S. Pat. No. 5,641,814); in anti-fungalcompositions (U.S. Pat. No. 5,663,208); in acne healing compositions(U.S. Pat. No. 5,646,190); in anti-inflammatory compositions (U.S. Pat.No. 5,648,380); in dermatological compositions (U.S. Pat. No.5,602,183); in sunscreen compositions (U.S. Pat. No. 5,674,912); inantihistamine compositions (U.S. Pat. No. 5,614,561); in cytoprotectivecompositions (U.S. Pat. No. 5,633,285); in wound healing compositionaffixed to razor cartridges (U.S. Pat. No. 5,682,302); and inregenerating compositions (EP 0 573 465 B1). However, none of thesepatents discloses or suggests the use of TRIAD as neuroprotective agent.

[0007] In view of the above, it is clear that there is a need for anamphiphilic antioxidant composition comprising pyruvate, antioxidant,and lipid(s) such as fatty acids to protect neuronal cells againstoxidant species.

[0008] The purpose of this invention is to fulfil this need along withother needs that will be apparent to those skilled in the art uponreading the following specification.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a neuroprotective compositionand more particularly to an amphiphilic antioxidative composition andits uses.

[0010] According to an aspect of the invention, the neuroprotectivecomposition comprises a therapeutically effective amount of a mixture ofpyruvate, antioxidant(s), and lipid(s) such as fatty acids. Thesecomponents are present in an amount that have a synergistic protectiveeffect on neuronal cells.

[0011] In a preferred embodiment, lipids consist of a mixture ofsaturated and unsaturated fatty acids selected from the group consistingof monogylcerides, digylcerides, trigylcerides, free fatty acids, andmixtures thereof.

[0012] Preferably, pyruvate is selected from the group consisting ofpyruvic acid, pharmaceutically acceptable salts of pyruvic acid,prodrugs of pyruvic acid, and mixtures thereof.

[0013] Preferably, also the antioxidant is selected from lipid-solubleantioxidants, and more preferably the antioxidant is selected from thegroup consisting of Vitamin A, carotene, Vitamin E, pharmaceuticallyacceptable salts thereof, and mixtures thereof.

[0014] According to an other aspect of the invention, theneuroprotective composition is used as such or as an active agent in thepreparation of a medication for the treatment of neuronal cells. Suchtreatments include the treatment brain trauma, brain or cerebrovascularischemia, neurodegenerative diseases, poisoning of neuronal cells, thediminution of drugs side effects and for preservation of neuronalgrafts.

[0015] According to an other aspect of the invention, the inventionprovides a method for treating neuronal oxidative stress relatedcondition, the method comprising administrating to a patient in needthereof a therapeutically effective amount of an antioxidativecomposition comprising pyruvate, at least one antioxidant and at leastone lipid.

[0016] Alternatively, the invention also provides a method for treatingneuronal oxidative stress related condition comprising: a)administrating to a patient in need thereof, a therapeutically effectiveamount of an antioxidative composition comprising pyruvate and at leastone antioxidant; and b) providing, into the blood circulation of thispatient, at least one lipid having a synergistic therapeutic effect onneuronal cells in combination with said antioxidative composition. Thelipid(s) could be provided to the patient by increasing its lipidicblood level ratio through its diet. Examples of neuronal oxidativestress related condition include a neurodegenerative disease, such asamyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's diseaseHuntington's disease, etc, brain trauma, brain or cerebrovascularischemia, neuronal cells poisoning, side effects caused by a drug andthe preservation of neuronal grafts.

[0017] According to an other aspect of the invention it is provided amethod for preparing a neuroprotective composition, the methodcomprising the steps of:

[0018] a) providing a therapeutically effective amount of: i) pyruvate,ii) at least one antioxidant; and iii) at least one lipid;

[0019] b) mixing together the components i), ii) and iii) of step a) ina physiological buffered saline solution to obtain a pharmaceuticallyacceptable homologous suspension; and optionally

[0020] c) centrifuging or filtering the homologous suspension obtainedin step b).

[0021] The buffered saline solution may comprises sodium, potassium,magnesium and calcium ions at physiological concentrations and ifnecessary, an emulsifier.

[0022] An advantage of the present invention is that it provideseffective means for preventing the loss of viability or functions ofneuronal cells in conditions of oxidative stress. It can also protect aneuronal cell from a toxic substance, stabilizes the cellular membraneof a neuronal cell and/or helps in the normalization of neuronalcellular functions.

[0023] Other objects and advantages of the present invention will beapparent upon reading the following non-restrictive description ofseveral preferred embodiments made with reference to the accompanyingdrawings.

DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is a graph showing the in vitro ROS production byperoxide-based prooxidant systems used with P19 neurons.

[0025] FIG. 2 is a graph showing the protection provided by TRIAD to P19neurons exposed for different times to XA/XAO mediated oxidative stress.

[0026] FIG. 3 is a graph showing the protection provided by TRIADcomponents to P19 neurons exposed for different times to XA/XAO mediatedoxidative stress.

[0027] FIG. 4 is a graph showing the protection provided by TRIAD to P19neurons exposed to oxidative stress mediated by different concentrationsof XAO.

[0028] FIG. 5 is a graph showing the protection provided by TRIAD to P19neurons exposed to hydrogen peroxide mediated oxidative stress.

[0029] FIG. 6 is a graph showing the protection provided by TRIADcomponents to P19 neurons exposed to hydrogen peroxide mediatedoxidative stress.

[0030] FIG. 7 is a graph showing the protection provided by TRIAD to P19neurons exposed to H₂O₂/Fe²⁺ prooxidant system.

[0031] FIG. 8 is a graph showing the protection provided by TRIADcomponents to P19 neurons exposed to H₂O₂/Fe²⁺ prooxidant system.

[0032] FIG. 9 is a graph showing the in vitro antioxidant capacity ofTRIAD in the H₂O₂ prooxidant system used with P19 neurons.

[0033] FIG. 10 is a graph showing the in vitro antioxidant capacity ofTRIAD in the conditions of H₂O₂/Fe²⁺ prooxidant system used with P19neurons.

[0034] FIG. 11 is a graph showing the in vitro antioxidant capacity ofTRIAD components in the conditions of H₂O₂ prooxidant system used withP19 neurons.

[0035] FIG. 12 is a graph showing the in vitro antioxidant capacity ofTRIAD components in the conditions of H₂O₂/Fe²⁺ prooxidant system usedwith P19 neurons.

DETAILED DESCRIPTION OF THE INVENTION

[0036] As stated hereinbefore the present invention relates to the useof an amphiphilic antioxidant compositions as neuroprotective agent. Asdisclosed herein, a composition comprising sodium pyruvate, antioxidantand lipid(s) such as fatty acids have neuroprotective actions againstoxidative stress.

[0037] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one ordinaryskilled in the art to which this invention belongs.

[0038] As used herein, the term “neuroprotective agent” or“neuroprotective composition” refers to any compound (or to any mixtureof compounds) that protects a neuronal cell from a toxic substance,stabilizes the cell membrane of a neuronal cell and/or helps in thenormalization of neuronal cell functions. A “neuroprotective agent”thereby prevents the loss of viability or functions of neuronal cells instressing conditions.

[0039] Therefore, the term “neuroprotection” as used herein refers tothe capacity of a neuroprotective agent to maintain or stimulate thecapacity of neuronal cells to maintain or recover their neuronalfunctions even in pathological or harmful conditions such as oxidativestress conditions.

[0040] As stated out above, the neuroprotective composition of theinvention comprises a mixture of (a) pyruvate, (b) at least oneantioxidant, and (c) at least one lipid such as fatty acids, preferablya mixture of saturated and unsaturated fatty acids. According to theinvention, these three components have a synergistic beneficial effecton neuronal cells, i.e. their combined effect is greater than the sum oftheir individual effects.

[0041] The pyruvate in the present invention may be selected from thegroup consisting of pyruvic acid, pharmaceutically acceptable salts ofpyruvic acid, prodrugs of pyruvic acid, and mixtures thereof. Ingeneral, the pharmaceutically acceptable salts of pyruvic acid may bealkali salts and alkaline earth salts. Preferably, the pyruvate isselected from the group consisting of pyruvic acid, lithium pyruvate,sodium pyruvate, potassium pyruvate, magnesium pyruvate, calciumpyruvate, zinc pyruvate, manganese pyruvate, methylpyruvate,α-ketoglutaric acid, and mixtures thereof. More preferably, the pyruvateis selected from the group of salts consisting of sodium pyruvate,potassium pyruvate, magnesium pyruvate, calcium pyruvate, zinc pyruvate,manganese pyruvate, and the like, and mixtures thereof. Most preferably,the pyruvate is sodium pyruvate.

[0042] The amount of pyruvate present in the neuroprotective compositionof the present invention is a therapeutically effective amount. Atherapeutically effective amount of pyruvate is that amount of pyruvatenecessary for the neuroprotective composition to prevent and/or reduceinjury of a neuronal mammalian cell. The exact amount of pyruvate willvary according to factors such as the type of condition being treated aswell as the other ingredients in the composition. Typically, the amountof pyruvate should vary from about 0.01 mM to about 100 mM. In apreferred embodiment, pyruvate is present in the composition of theneuroprotective extracellular medium in an amount from about 0.1 mM toabout 30 mM, preferably from about 0.5 mM to about 10 mM. In the mostpreferred embodiment, the neuroprotective composition comprises about 10mM of sodium pyruvate.

[0043] Antioxidants, including vitamin antioxidants, are substanceswhich inhibit oxidation or suppress reactions promoted by oxygen, oxygenfree radicals (OFR), oxygen reactive species (ORS) including peroxides.Antioxidants, especially lipid-soluble antioxidants, can be absorbedinto the cell membrane to neutralize oxygen radicals and thereby protectthe membrane. The antioxidants useful in the present invention arepreferably vitamin antioxidants that may be selected from the groupconsisting of all forms of Vitamin A including retinal and3,4-didehydroretinal, all forms of carotene such as alpha-carotene,p-carotene, gamma-carotene, delta-carotene, all forms of Vitamin C(D-ascorbic acid, L-ascorbic acid), all forms of tocopherol such asVitamin E (Alpha-tocopherol,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-decyl)-2H-1-benzopyran-6-ol),β-tocopherol, gamma-tocopherol, delta-tocopherol, tocoquinone,tocotrienol, and Vitamin E esters which readily undergo hydrolysis toVitamin E such as Vitamin E acetate and Vitamin E succinate, andpharmaceutically acceptable Vitamin E salts such as Vitamin E phosphate,prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E,pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, andVitamin E, and the like, and mixtures thereof. Preferably, theantioxidant is selected from the group of lipid-soluble antioxidantsconsisting of Vitamin A, p-carotene, Vitamin E, Vitamin E acetate, andmixtures thereof. More preferably, the antioxidant is Vitamin E orVitamin E acetate. Most preferably, the antioxidant is Vitamin Eacetate. Analogues of Vitamin E such as Trolox®, a compound which ismore hydrosoluble than natural forms of Vitamin E and which could reachintracellular sites more rapidly, could also be used according to thepresent invention.

[0044] The amount of antioxidant present in the neuroprotectivecomposition of the present invention is a therapeutically effectiveamount. A therapeutically effective amount of antioxidant is that amountof antioxidant necessary for the neuroprotective composition to preventand/or reduce injury of a neuronal mammalian cell. The exact amount ofantioxidant will vary according to factors such as the type of conditionbeing treated as well as the other ingredients in the composition.Typically, the amount of antioxidant should vary from about 0.01 unit/mlto about 10 unit/ml. In a preferred embodiment, vitamin E antioxidant ispresent in the composition of the neuroprotective extracellular mediumin an amount from about 0.01 unit/ml to about 10 unit/ml, preferablyfrom about 0.05 to about 5 unit/ml. In the most preferred embodiment,the neuroprotective composition comprises about 1 unit of antioxidantα-tocopherol type VI in oil) per ml of neuroprotective composition.

[0045] As it is well known, lipids are esters or carboxylic acidcompounds found in animal and vegetable fats and oils. The compositionmay comprises a single type of lipid or various types of differentlipids. Preferably lipids are in the form of a mixture of saturated andunsaturated fatty acids. However, other types of lipids could be usedsuch as glycolipids and phospholipids (e.g. lecithin). Lipid(s) ormixture thereof are selected among those lipids required for thestabilization and/or repair of the membrane of neuronal mammalian cells.These lipids may be derived from animal or vegetables. In a preferredembodiment, selected lipids are in the form of mono-, di-, ortriglycerides, or free fatty acids, or mixtures thereof, which arereadily available for the stabilization or repair of the membrane ofneuronal mammalian cells. Artificial lipids which are soluble in organicsolvents and are of a structural type which includes fatty acids andtheir esters, cholesterols, cholesteryls esters could also be usedaccording to the present invention.

[0046] In a more preferred embodiment, the saturated and unsaturatedfatty acids are those deriving from egg yolk. According to the use ofthe neuroprotective compositions of the invention, replacing egg yolk asa source of fatty acids by chemical preparations of unsaturated,polyunsaturated and/or saturated fatty acids compatible with, and inproportions similar to those found in cell membranes may be advantageousor reveal necessary to insure a controllable quality of preparations.

[0047] The amount of lipid(s) such as fatty acids present in theneuroprotective composition of the present invention is atherapeutically effective amount. A therapeutically effective amount offatty acids for instance is that amount of fatty acids necessary for theneuroprotective composition to prevent and/or reduce injury of aneuronal mammalian cells. The exact amount of lipid(s) or fatty acidswill vary according to factors such as the type of condition beingtreated as well as the other ingredients in the composition. Typically,the amount of lipid(s) or fatty acids should vary from about 0.001% v/vto about 1% v/v. In a preferred embodiment, fatty acids are present inthe neuroprotective composition in an amount from about 0.001% v/v toabout 0.3% v/v, preferably from about 0.005% v/v to about 0.1% v/v. Inthe most preferred embodiment, the neuroprotective composition comprisesabout 0.1% v/v of fresh egg yolk.

[0048] As the lipidic blood level of an individual is normally about0.5-0.6% of the total serum volume, the lipidic portion could be omittedfrom the neuroprotective composition of the invention. It could bepossible to provide into the blood circulation of this individual atleast one lipid having a synergistic therapeutic effect on neuronalcells with the others component of the antioxidative neuroprotectivecomposition of the invention. For instance, selected lipid(s) could beprovided by increasing the lipidic blood level ratio of this individualthrough the diet. Lipids which could have a synergistic therapeuticeffect without being harmful to a patient could be selected from thegroup consisting of phospholipids, glycolipids, fatty acids, and mixturethereof.

[0049] Further agents can be joint to the neuroprotective composition ofthe invention. For examples various antioxidants may complete the actionof neuroprotective composition such as:

[0050] ceruloplasmin or its analogues since it can scavenge *O₂ ⁻radicals and has a ferroxidase activity which oxidizes Fe²⁺ to Fe³⁺;

[0051] metal chelators/scavengers (e.g. desferrioxamine [Desferal®], asmall substance capable to scavenge Fe³⁺ and other metal ions);

[0052] proteins or their fragments that can bind metal ions such asferritin or transferrin which both bind Fe³⁺;

[0053] scavengers of *OH (hydroxyl) or NO (nitric oxide) radicals (e.g.mannitol).

[0054] small scavengers of *O₂ ⁻(superoxide), *OH (hydroxyl) or NO(nitric oxide) radicals (e.g. acetyl salicylic acid, scavenger of *O₂ ⁻;mannitol or captopril, scavengers of *OH) or molecules that inhibit thegeneration of these radicals (e.g. arginine derivatives, inhibitors ofnitric oxide synthase which produce NO);

[0055] proteins or their fragments that scavenge oxygen free radicalsand can assist the protective action of ceruloplasmin (e.g. superoxidedismutase which dismutate *O₂ ⁻; hemoglobin which traps NO); and

[0056] proteins or their fragments that can scavenge H₂O₂ (hydrogenperoxide) in cases where they may exert a more potent or durableprotective action than pyruvate (e.g. catalase, glutathion peroxidase).

[0057] The composition of the invention may also comprises modulators ofbrain functions such as neurotransmitters, neuropeptides, hormones,trophic factors, or analogs of these substances that act by binding tobrain receptors (e.g. DOPA in Parkinson's disease).

[0058] Further to the therapeutic agents, the neuroprotectivecomposition of the invention may also contain preserving agents,solubilizing agents, stabilizing agents, wetting agents, emulsifiers,sweeteners, colorants, odorants, salts, buffers, or coating agents. Forpreparing the neuroprotective composition, methods well known in the artmay be used.

[0059] The method of preparation of the neuroprotective compositions ofthe invention is very simple as it consists simply in the mixing ofcomponents in a buffered saline solution in order to get a homogenoussuspension. Suitable saline solution comprises sodium, potassium,magnesium and calcium ions at physiological concentrations, has anosmotic pressure varying from 280 to 340 mosmol, and a pH varying from7.2 to 7.4. Depending of the amount and of type of lipid(s) which isused, the saline may also comprises an emulsifier. Preferably, thebuffered saline solution is selected from the group consisting ofmodified Krebs-Henseleit buffer (KH) and phosphate buffer saline (PBS),both at pH 7.4. The homogenous suspension obtained can further becentrifuged and/or filtered to reduce its viscosity and/or eliminatednon-soluble particles.

[0060] Obviously, this simple method can be modified according to theuse of the neuroprotective composition. In the example found hereunder,a genuine preparation was used. Centrifuged and/or filtered preparationscould also have been used. However, it is important to note thatmodifications in the modality of preparation can influence the resultingeffects of the neuroprotective composition. For example, varying the pHof the composition (or buffer) can slightly modify the ionization stateof carboxylic functions of pyruvate and thus alter its solubility and/orreaction with H₂O₂, while the dialysis of the composition would reducethe amount of pyruvate in the final preparation, unless it is donebefore addition of pyruvate. A person skilled in the art will know howto adapt the preparation of the neuroprotective composition of theinvention according to its desired use in specific conditions in orderto obtain positive desired effects.

[0061] The neuroprotective composition of the invention could besuitable to treat diseases and pathological conditions such as braintrauma and diseases which were shown to involve oxidative stressconditions such as amyotrophic lateral sclerosis and neurodegenerativeParkinson's, Alzheimer's and Huntington's diseases. Theseneuroprotective compositions could also be involved in the treatment ofpoisoning or diminution of side effects of various drugs (such aschemotherapeutic and immunosuppressive drugs) to the brain and/or toneuronal cells. Indeed, deleterious action of various toxicants anddrugs is exerted via production of ROS.

[0062] The neuroprotective composition of the invention has potentialapplications in both fast (in minutes; especially due to the pyruvate)and long term treatments (hours and days; due to the antioxidant andlipid(s) such as fatty acids). The amount to be administered is atherapeutically effective amount. A therapeutically effective amount ofa neuroprotective composition is that amount necessary for protecting aneuronal cell from the loss of viability or function induced by a toxicsubstance, stabilizing the cell membrane of neuronal cells and/orhelping in the normalization of neuronal cell functions. Suitabledosages will vary, depending upon factors such as the type and theamount of each of the components in the composition, the desired effect(fast or long-term), the disease or disorder to be treated, the route ofadministration and the age and weight of the individual to be treated.

[0063] The neuroprotective composition of the invention and/or morecomplex pharmaceutical compositions comprising the same may be givenorally (per os) in the form of tablets, capsules, powders, syrups, etc.since all their components are absorbable by the gastrointestinal tract.Others administration ways can also be considered (rectal and vaginalcapsules or nasally by means of a spray). They may also be formulated ascreams or ointments for topical administration. They may also be givenparenterally, for example intravenously, intramuscularly orsub-cutaneously by injection or by infusion. Intravenous administrationcan be a way for fast answer in various clinical conditions (e.g.ischemic brain, -brain trauma, stroke and heart attacks, post-surgerytreatments, etc). Obviously, the neuroprotective compositions of theinvention may be administered alone or as part of a more complexpharmaceutical composition according to the desired use and route ofadministration. Anyhow, for preparing such compositions, methods wellknown in the art may be used.

[0064] As it will now be demonstrated by way of an example hereinafter,the composition of the invention possesses a strong neuroprotectiveactivity i.e. the capacity to maintain the viability and functions ofneurons at their normal level or to induce a fast recovery to the normallevel, even in pathological or harmful conditions such as oxidativestress conditions. These conditions can occur at post-ischemiareperfusion of the brain associated with an attack to brain vasculature,cerebral trauma or a heart stroke/attack, in various neurodegenerativediseases, in epilepsy, following an exposure to neurotoxicants, or asside-effects of drugs and inflammation. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

EXAMPLE: Neuroprotective Actions of TRIAD Against Oxidative StressAbstract

[0065] This work shows that TRIAD, a combination of sodium pyruvate,vitamin E and egg yolk fatty acids, has an antioxidant protective actionon cultured P19 neurons exposed to oxidative stress. Oxidative stresswas induced by incubation with prooxidant systems that generate majorreactive oxygen species produced by ischemia-reperfusion of the brain invivo, namely 1) xanthine/xanthine oxidase system to produce .O₂ ⁻superoxide radicals and H₂O₂, 2) H₂O₂ it self, and 3) H₂O₂ in thepresence of Fe²⁺ to produce .OH hydroxyl radicals. TRIAD-inducedresistance to injury caused by oxidative stress was assessed bymeasurement of cell viability. TRIAD concentrations less than 3×permitted to achieve complete protection of neurons. Optimalconcentrations of TRIAD with neurons exposed to peroxide-based systemswere directly related to the oxidant power of the systems as measured byoxidation of N,N-diethyl-p-phenylenediamine. However, higherconcentrations of TRIAD than those predicted from in vitro analyses wererequired to protect neurons against oxidative stress. In addition, theresults also show that the respective contribution of pyruvate and offatty acids+vitamin E combination may differ between prooxidant systemsand between in vitro or cell culture situations. These results indicatethat TRIAD components have different mechanisms of action and that thesemechanisms are further modulated by cell metabolism. Generally, in ourexperimental models, pyruvate was a major contributor of the antioxidantaction of TRIAD and its effect was increased by fatty acids and vitaminE in some cases in an additive manner and in other casessynergistically.

[0066] Abbreviations DPD: N,N-diethyl-p-phenylenediamine; KH:Krebs-Henseleit; LD₅₀: lethal dose 50 or dose that causes 50% mortality;PBS:phosphate buffer saline; OFR: oxygen free radical; ROS: reactiveoxygen species; XA: xanthine; XAO: xanthine oxidase, SOD: superoxidedismutase; CAT: catalase; GP: glutathion (GSH)-peroxidase.

1. Introduction

[0067] 1.1 Oxidative Stress and Antioxidant Defenses in Normal andPathophysiological Heart and Brain

[0068] Reactive oxygen species (ROS) including hydrogen peroxide, oxygenfree radicals (OFR) such as superoxide and hydroxyl radicals, and theirderivatives are generated by normal cellular metabolism but are potentcellular toxicants when they are produced in excess and thus cause anoxidative stress to cells (LeBel and Bondy, 1991; Gutteridge, 1994;Chan, 1996). The organism has several strategies to maintain ROS-induceddamage at low levels: a) to eliminate ROS (e.g. SOD, CAT and GP enzymesshown in FIG. 1), b) to scavenge ROS by trapping them (e.g. ascorbicacid) or by breaking their propagation (e.g. vitamin E), c) to sequesteriron or other metals in non- or low reactive forms, and d) to repairmolecular damages (Gutteridge, 1994).

[0069] ROS have been implicated in the development of many heart andbrain dysfunctions (Takemura et al., 1994; Chan, 1996; Maiese, 1998) andischemia/reperfusion insults to these organs are among the leadingcauses of mortality in America (Takemura et al., 1994; Chan, 1996;Maiese, 1998). These insults are caused by complete or partial localocclusions of vasculature and by trauma to heart and brain. ROS as thosefound in ischemia-reperfusion events are also involved in the evolutionof several neurodegenerative diseases or produced in brain following anexaggerated activity of this organ (e.g. epilepsy).

[0070] Various pathways generating superoxide radical (.O₂ ⁻) and otherROS—also known as reactive oxygen intermediates (ROI)— have beenidentified, such as: activation of polymorphonuclear leukocytes,autoxidation of catecholamines, reactions of xanthine oxidase and NADPHoxidase, or metabolism of arachidonic acid. The harmful effects ofsuperoxide radical and its by-products are dramatically increased in thepresence of transition metals. The ferrous (Fe²⁺) ion generated by theHaber-Weiss reaction catalyses the formation of the highly aggressivehydroxyl (—OH) radical, via Fenton reaction. The OFR concentration atreperfusion is higher than during ischemia. OFR may contribute toreperfusion injury by interacting with membrane polyunsaturated fattyacids (PUFA) and generating lipid peroxides which increase membranepermeability and alter ionic homeostasis. Inhibition of free radicalaccumulation with OFR scavengers, antioxidant enzymes, and spin-trapagents was shown to reduce the severity of damages to brain. However,the benefits of these treatments gradually vanish with time, especiallyduring long-term utilization in neurodegenerative diseases, or arelessen by the apparition of adverse secondary effects. Therefore, theidentification of other therapeutics agents still remains highlydesirable.

[0071] 1.2 Aspects on TRIAD and its Therapeutic Role

[0072] As stated herein before, TRIAD is a combination of pyruvate,antioxidant and fatty acids for which many uses have been patented.Preferably, TRIAD comprises sodium pyruvate, vitamin E and egg yolk.Although this combination is also known under the name of CRT (CellularResuscitation Therapy), the current denomination of TRIAD is usethroughout this document.

[0073] These three agents were shown to act synergistically toameliorate wound healing (Martin, 1996; Sheridan et al., 1997) and toreduce oxidative damage to keratinocytes and monocytes exposed toultraviolet light (Martin, 1996) or to hepatocytes treated withdoxorubicin (Gokhale et al., 1997). The presumed respective role of eachagent of the antioxidant combination is a) for pyruvate, to bindstochiometrically to H₂O₂, b) for vitamin E, to interrupt thepropagation of lipid peroxidation, and c) for egg yolk, to provide abalanced mix of fresh unsaturated and saturated fatty acids which willhelp in membrane repair (Martin, 1996).

[0074] 1.3 Presentation of the Study

[0075] The goal of this study was to determine if TRIAD has anantioxidant protective action on cultured P19 neurons exposed tooxidative stress. The choice of this model is related to the fact thatthe P19 cell line is establishing itself as a flexible model of neuronsof central nervous system. Oxidative stress was induced by incubationwith prooxidant systems that generate major ROS produced byischemia-reperfusion in vivo. Prooxidant systems used are: i) XA/XAOsystem to produce .O₂— and H₂O₂ ii) H₂O₂ it self, and iii) H₂O₂ in thepresence of Fe²⁺ to produce .OH. Resistance of neurons to injury inducedby oxidative stress was assessed by measurement of cell viability. Inall cases, different concentrations of TRIAD were tested in order todetermine those that permitted to achieve a complete protection and alsotested the contribution of TRIAD components to the overall protection.In addition, when applicable, the antioxidant properties of TRIAD invitro were measured in order to understand some aspects of theprotection afforded by this mix in live models.

2. Materials and Methods

[0076] Materials

[0077] Vitamin E (α-tocopherol type VI in oil), sodium pyruvate,ethylenediamine tetraacetic acid (EDTA), N,N-diethyl-p-phenylenediamine(DPD), and xanthine (XA) were purchased from Sigma Chem. Co. Xanthineoxidase (XAO) was from Boehringer Mannheim. Neurobasal®, L-glutamine andB27 supplement were from Gibco-BRL. Alamar Blue was purchased fromMedicorp (Montreal, Quebec). Fresh egg yolk was used as the source offatty acids. The other current chemicals were reagent grade (from SigmaChem. Co., St-Louis) and were used without further purification.

[0078] Methods

[0079] 2.1 Preparation of TRIAD

[0080] The 1× TRIAD concentration was prepared as per Gokhale et al.(1997) and contained 0.1% v/v fresh egg yolk, 1 unit/ml vitamin E(α-tocopherol type VI in oil) and 10 mM sodium pyruvate. Stock 5× (5fold) or 10× (10 fold) concentration of TRIAD was freshly preparedbefore each experiment by carefully mixing the three agents to get ahomogenous suspension. TRIAD mixtures were made in phosphate buffersaline (PBS; 136 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄ and 8 mM Na₂HPO₄, pH7.4). Pyruvate was soluble in and egg yolk miscible with this salinephysiological buffer. Aseptically drawn egg yolk and vitamin Esuspension (vitamin E in oil combined to 70% ethanol in a 2.5:1 ratio)were added at the desired final concentrations to a 100 mM stockpyruvate solution prepared in PBS and filter-sterilized on 0.22 μm.

[0081] Although not tested with neuronal cells, a modified preparationof TRIAD was shown to be effective to protect isolated hearts.Modification of TRIAD preparations was as follows: 5× or 10× genuinepreparations were centrifuged at 15000×g for 20 min, at 4° C., and theresulting supernatants (S1) filtered on Whatman paper filter #54. Thefinal filtered supernatant was named TRIAD (S2) and used to perfusehearts. The different concentrations of TRIAD (S2) preparation wereobtained by subsequent dilution with Krebs-Henseleit physiologicalsaline buffer (i.e. TRIAD (S2) 1× was obtained by 10 fold dilution ofstock TRIAD (S2) 10× preparation).

[0082] 2.2 Culture and Neuronal Differentiation of P19 Cells

[0083] Culture and neuronal differentiation of P19 embryonal carcinomacells were done according to the procedures of Jeannotte et al. (1997)with the following modifications for microscale adaptation of culturesto 96-well plates: cell aggregates obtained at day 4 of the treatment ofP19 cells with retinoic acid were trypsinized with 0.025% trypsin-1 mMEDTA in PBS and subjected to mechanical passages to obtain individualcells which were seeded in gelatin-precoated microwells at a density of0.7-1×10⁵ cells per well. The newly seeded cells (neurons) were culturedin supplemented Neurobasal medium (Neurobasal® containing 0.5 mML-glutamine and 0.5% B27 supplement) until exposure to oxidativeconditions at day 7. Because this defined serum-free medium sustainsgrowth of P19 neurons (Yao et al., 1995) but discourages theproliferation of fibroblasts, another cell derivative of thedifferentiation of P19 cells with retinoic acid (McBurney, 1993;Jeannotte et al., 1997), the cell populations in microwells werecomposed mostly of neurons (>95%).

[0084] 2.3 Exposure of P19 Neurons to Prooxidant Systems

[0085] The prooxidant systems tested with P19 neurons were XA/XAO, H₂O₂,and H₂O₂/Fe⁺². Before neurons were exposed to either one of thesesystems, they were carefully washed with PBS and then incubated at 37°C., in an atmosphere of 95% ambient air and 5% CO₂, and in the specificconditions of each system, as follows: i) from 0 to 90 min in thepresence of PBS containing 500 μM XA and 50 mU/ml XAO, with the enzymeadded last to start the reaction; ii) for 30 min in the presence of PBScontaining 0 to 10 mM H₂O₂ with peroxide added last; and iii) for 30 minin the presence of PBS containing 0 to 10 mM H₂O₂ plus 50 μM FeCl₂, withperoxide added last. Conditions were initially taken from Cini et al.(1994) for XA/XAO, from Desagher et al. (1996) for H₂O₂, and fromTakemura et al. (1994) for H₂O₂/Fe⁺² systems respectively, and adaptedto produce dose- or time-dependent cell mortality in P19 neurons. Thefirst studies done with the H₂O₂/Fe system included the addition of 500μM ascorbic acid; however, since responses of P19 neurons were similarwhether or not ascorbic acid was present, this vitamin was omitted insubsequent experiments.

[0086] When TRIAD or its components were tested for their antioxidantaction, they were administered to cells just prior the addition of XAOor H₂O₂. After incubation of cells under oxidative conditions, theprooxidant medium was removed and replaced with 200 μl of supplementedNeurobasal-minus AO (Neurobasal® containing L-glutamine and theB27-minus AO supplement). B27-minus AO is a version of B27 supplementsold by Gibco-BRL from which normally present antioxidants (AO=vitaminE, catalase, SOD and GSH) have been removed. Cells were incubated inthis Neurobasal-minus AO medium for 16 h, at 37° C., 5% CO₂, and for afurther 7 h in the same medium but in the presence of Alamar Blue forviability measurement.

[0087] Another protocol was also tested for XA/XAO system to generate amilder oxidative stress. In this new protocol, P19 neurons were exposedduring 40 min to 250 μM XA and varying concentrations of XAO (0 to 20mU/mL) in PBS. At end of stress, PBS was changed for Neurobasal-minus AOmedium and cells were incubated for 7 h, readily in the presence ofAlamar Blue, for viability measurement.

[0088] 2.4 Cell Viability Assay

[0089] Fifteen (15) μl Alamar Blue was added to the culture medium ofeach well (200 μl) and incubation resumed for 7 h at 37° C., 5% CO₂. A180-μl aliquot of each culture medium was read by fluorescence using awavelength of 544 nm for excitation and of 590 nm for emission;fluorescence increases upon reduction of the dye by metabolic activityof viable cells. Fluorescence determinations were done with afluorimeter adapted to read microplates. Viability is reported as %,comparing the fluorescence units obtained for cells exposed to oxidativeconditions to those of control (non-exposed) cells.

[0090] 2.5 In Vitro Antioxidant Capacity

[0091] Oxidation of N,N-diethyl-p-phenylenediamine (DPD) by a prooxidantsystem was used as a general reporter of the amount of ROS generated bythat system (Anonymous, 1985; Chahine et al., 1991). Antioxidantcapacity of preparations of TRIAD (or of its components) was defined asthe ability to inhibit the oxidation of DPD by prooxidants.

[0092] To estimate the antioxidant capacity of TRIAD preparations in theprooxidant conditions used with P19 neurons, DPD was added to a finalconcentration of 32 mM to 200 μl of each prooxidant system describedabove (see section 2.4) and incubated for the times tested with thecells. At the end of incubation, the amount of oxidized DPD wasdetermined at 560 nm using a spectrophotometer adapted to microscalemeasurement.

3. Results

[0093] The P19 embryonal carcinoma cell line is establishing its placeas a versatile cell model for neurons of central nervous system(McBurney, 1993; Yao et al., 1994; Finley et al., 1996; Parnas andLinial, 1997; Jeannotte et al., 1997). These cells differentiate intoneurons, astrocytes and fibroblast-like cells following induction withretinoic acid, and their neuronal derivatives mature into functionalneurons. Indeed, P19-derived neurons express a variety ofneuron-specific proteins, acquire cell polarity of neurons, formsynapses, synthesize and release neurotransmitters and neuropeptides,and their membranes respond to electrophysiological stimuli (McBurney,1993; Finley et al., 1996; Parnas and Linial, 1997; Jeannotte et al.,1997). The P19 system presents several advantages over other neuronalmodels for screening tests using cultured cells: i) P19 neurons likeprimary neurons are highly differentiated (in contrast, neuroblastomacells often used as neuronal models are poorly differentiated), ii)acquisition of P19 neurons does not depend on the sacrifice of animals,and iii) although P19 neurons are post-mitotic and therefore do notdivide as do neuroblastoma cells, they can be obtained in largequantities since P19 stem cells propagate at high rates. Consideringthat they resemble primary neurons and can be easily and reproductivelyobtained, we thus used P19 neurons to study the neuroprotective actionof TRIAD.

[0094] Legends to the Figures

[0095] FIG. 1. In vitro ROS production by peroxide-based prooxidantsystems used with P19 neurons. The relative amount of ROS produced byH₂O₂ and H₂O₂/Fe²⁺ systems in the conditions used with P19 neurons weremeasured spectrophotometrically, in absence of cells, by followingoxidation of N,N-diethyl-p-phenylenediamine (DPD). The experiment wasdone twice, in triplicate determinations, and results are expressed asmeans±errors to the means. The H₂O₂/Fe²⁺ system contained 50 μM FeCl₂;the addition of 500 μM ascorbic acid to this system did not change theamount of oxidized DPD (not shown), indicating that iron ions were in aconcentration sufficient to increase the oxidative stress induced byH₂O₂ alone.

[0096] FIG. 2. Protection provided by TRIAD to P19 neurons exposed fordifferent times to XA/XAO mediated oxidative stress. P19 neurons wereexposed from 0 to 90 min to 500 μM XA and 50 mU/mL XAO in the absence(No protection) or presence of different concentrations of TRIAD. At endof stress, cells were incubated for 16 h in a fresh provision of culturemedium lacking XA, XAO and TRIAD. Afterward, Alamar Blue was added andcells were further incubated for 7 h for viability determination.Viability values are reported as percentages, with 100% corresponding tothe response of P19 neurons not exposed to XA/XAO. The experiment wasdone once, in duplicate determinations, and results are expressed asmeans (±errors to the means). Error bars are similar for all curvesalthough they are shown for two curves only, for the purpose of clarity.

[0097] FIG. 3. Protection provided by TRIAD components to P19 neuronsexposed for different times to XA/XAO mediated oxidative stress.Exposure to XA/XAO and viability measurement were done as indicated inthe legend to FIG. 2. The experiment was done twice, in duplicatedeterminations, and results are expressed as means±errors to themeans.Vit.E, vitamin E; F.A., fatty acids.

[0098] FIG. 4. Protection provided by TRIAD to P19 neurons exposed tooxidative stress mediated by different concentrations of XAO. P19neurons were exposed for 40 min to 250 μM XA and differentconcentrations of XAO in the absence (No protection) or presence of 0.5×TRIAD. At end of stress, cells were incubated for 7 h in a freshprovision of culture medium lacking XA, XAO and TRIAD, but containingAlamar Blue for viability determination. Viability values are reportedas percentages, with 100% corresponding to the response of P19 neuronsnot exposed to XA/XAO. The experiment was done twice, in triplicatedeterminations, and results are expressed as means±errors to the means.

[0099] FIG. 5. Protection provided by TRIAD to P19 neurons exposed tohydrogen peroxide mediated oxidative stress. P19 neurons were exposedduring 30 min to different concentrations of H₂O₂ in the absence(control=no protection) or presence of different concentrations ofTRIAD. At end of stress, cells were incubated for 16 h in a freshprovision of culture medium lacking H₂O₂ and TRIAD. Alamar Blue was thenadded and cells were further incubated for 7 h for viabilitydetermination. Viability values are reported as percentages, with 100%corresponding to the response of P19 neurons not exposed to H₂O₂. Theexperiment was done 5 times, in duplicate determinations, and resultsare expressed as means±S.D. Omission of the 16 h incubation period didnot change the relative responses.

[0100] FIG. 6. Protection provided by TRIAD components to P19 neuronsexposed to hydrogen peroxide mediated oxidative stress. Exposure to H₂O₂and viability measurement were done as indicated in the legend to FIG.5. The experiment was done four times, in triplicate determinations, andresults are expressed as means±S.D. Vit.E, vitamin E; F.A., fatty acids.

[0101] FIG. 7. Protection provided by TRIAD to P19 neurons exposed toH₂O₂/Fe²⁺ prooxidant system. Cell treatments were as described in thelegend to FIG. 5 except that iron (50 μM FeCl₂) was present in thestress medium to generate hydroxyl radicals. Viability values arereported as percentages, with 100% corresponding to the response of P19neurons not exposed to H₂O₂/Fe²⁺. The experiment was done 3 times, induplicate determinations, and results are expressed as means±S.D.

[0102] FIG. 8. Protection provided by TRIAD components to P19 neuronsexposed to H₂O₂/Fe²⁺ prooxidant system. Exposure to H₂O₂/Fe²⁺ andviability measurement were done as indicated in the legend to FIG. 7.The experiment was done twice, in duplicate determinations, and resultsare expressed as means±errors to the means. Vitamin E+fatty acids didnot protect in that system (not shown).

[0103] FIG. 9. In vitro antioxidant capacity of TRIAD in the conditionsof H₂O₂ prooxidant system used with P19 neurons. Effect of differentconcentrations of TRIAD on the oxidation of DPD induced by H₂O₂. Theexperiment was done 5 times, in duplicate determinations, and resultsare expressed as means±S.D.

[0104] FIG. 10. In vitro antioxidant capacity of TRIAD in the conditionsof H₂O₂/Fe²⁺ prooxidant system used with P19 neurons. Effect ofdifferent concentrations 2+of TRIAD on the oxidation of DPD induced byH₂O₂/Fe . The experiment was done 7 times, in duplicate determinations,and results are expressed as means±S.D.

[0105] FIG. 11. In vitro antioxidant capacity of TRIAD components in theconditions of H₂O₂ prooxidant system used with P19 neurons. Effect ofTRIAD components on the oxidation of DPD induced by H₂O₂. The experimentwas done 4 times, in duplicate determinations, and results are expressedas means±S.D. Vit. E, vitamin E; F.A., fatty acids.

[0106] FIG. 12. In vitro antioxidant capacity of TRIAD components in theconditions of H₂O₂/Fe²⁺ prooxidant system used with P19 neurons. Effectof TRIAD 2+components on the oxidation of DPD induced by H₂O₂/Fe . Theexperiment was done 3 times, in duplicate determinations, and resultsare expressed as means±S.D. Vit. E, vitamin E; F.A., fatty acids.

[0107] 3.1 In Vitro Oxidant Capacity of Peroxide-Based ProoxidantSystems Used with P19 Neurons

[0108] Three prooxidant systems were used to induce oxidative stress inP19 neurons, namely XA/XAO, H₂O₂, and H₂O₂/Fe⁺². The relative oxidantcapacity of the two peroxide-based system was determined by followingoxidation of DPD in vitro. FIG. 1 shows that addition of Fe⁺² increasedthe oxidant power of H₂O₂. Comparison with the oxidant power of XA/XAOsystem could not be done since this system did not directly oxidize DPD.It is believed that addition of SOD enzyme to XA/XAO system in vitrowould convert .O₂ ⁻ radicals produced by the system to measurableproportionate amounts of H₂O₂ molecules.

[0109] 3.2 Neuroprotection Afforded by TRIAD Against Oxidative StressInduced by XA/XAO

[0110] When exposed during 90 min to TRIAD in the absence of anoxidative stress, P19 neurons remained completely viable even forconcentration 9× of the antioxidant mix (results not shown). Sincegenuine preparations of TRIAD were not toxic to these cells, they wereused without further treatment (i.e. TRIAD and not TRIAD (S2) was usedwith P19 neurons). However, as explained hereinbefore (see section 2.1),the genuine preparations could have been centrifuged and the resultingsupernatants filtered.

[0111] XA/XAO system was used to generate superoxide radicals (.O₂ ⁻) inaddition to H₂O₂. When exposed to that system for different times, alarge proportion of P19 neurons died after 5 min (FIG. 2, Noprotection). When TRIAD was present in the culture medium, it decreasedcell mortality caused by the prooxidant conditions. Protection wasconcentration dependent and reached almost completion at concentration9× of TRIAD (FIG. 2). The individual contribution of fatty acids,vitamin E and pyruvate to the protection provided by TRIAD againstneuronal death caused by XA/XAO was also determined. A 1× concentrationfor TRIAD and its components was used instead of an optimal value of 9×in order to reveal the eventual synergistic effects, if any. FIG. 3shows that pyruvate 1× protected only slightly less than TRIAD 1×. Fattyacids and vitamin E together protected similarly to pyruvate or TRIADduring the 30 first min of incubation with XA/XAO (FIG. 3). However, theprotective effect fell off rapidly from 30 to 90 min while that ofpyruvate remained stable (FIG. 3). At 90 min of exposure, the summationof the protective effect separately shown by pyruvate and by fattyacids+vitamin E reproduced the protection provided by TRIAD itself (FIG.3), indicating that fatty acids and vitamin E had an additive effect onpyruvate action.

[0112] It was realized that the pro-oxidant conditions used just abovewere probably very drastic since most neurons died after 5 min (FIGS. 2and 3, No protection). According to the protocol used, cells wereexposed to 500 μM XA and 50 mU/mL XAO for up to 90 min, then incubatedin fresh culture medium—not containing XA/XAO—for 16 h hours in theabsence of AlamarBlue, and for a further 7 h in the presence of the dye.It was believed that any residual amount of XA/XAO left in the culturemedium had time to continue to attack neurons, explaining why viabilitywas lost abruptly. Therefore, milder conditions were applied in order toobserve a gradual loss of viability. In the new protocol, neurons werefirst exposed for 40 min to 250 μM XAN in the presence of variousconcentrations of XAO (0-20 mU/mL), and then readily incubated in freshculture medium in the presence of AlamarBlue for 7 h. If there wasresidual amount of XAO left in the fresh medium, it increased mortalitybut in a manner proportional to the initial concentration of the enzyme.With the new protocol, the loss of viability caused by XA/XAO was indeedgradual and depended on the concentration of XAO in the solution (FIG.4). Interestingly, 0.5× TRIAD was sufficient to provide almost completeprotection in those conditions.

[0113] 3.3 Neuroprotection Afforded by TRIAD Against Oxidative StressInduced by H₂O₂

[0114] FIG. 5 shows that P19 neurons died in a concentration dependentmanner when exposed to hydrogen peroxide. The LD₅₀ value was 0.3 mMhydrogen peroxide. TRIAD protected cells against death caused by H₂O₂and a complete protection was achieved with 1× concentration of theantioxidant mix. TRIAD 3× and 5× also provided complete protection (notshown). When assayed at 0.5× suboptimal concentration of TRIAD, fattyacids or vitamin E alone did not provide protection against oxidativestress caused by hydrogen peroxide (not shown). Combination of theseagents was protective up to 0.5 mM H₂O₂ but fell off rapidly at higherconcentrations of the prooxidant (FIG. 6). In contrast, pyruvateprovided a substantial and stable protection up to 10 mM H₂O₂ (FIG. 6).Comparison of the protection separately provided by 0.5×pyruvate and0.5× TRIAD shows that 0.5× TRIAD was more efficient by about 2-fold(FIG. 6), indicating that fatty acids and vitamin E increased theprotective action of pyruvate in a synergistic manner.

[0115] 3.4 Neuroprotection Afforded by TRIAD Against Oxidative StressInduced by H₂O₂ in the Presence of Iron

[0116] Addition of Fe²⁺ to generate hydroxyl radicals was slightly moredeleterious to neurons than hydrogen peroxide alone. As an example, 1 mMH₂O₂ caused approximately 70% cell mortality (FIG. 5) whereas the sameconcentration of peroxide in the presence of iron caused more than 80%cell mortality (FIG. 7). This is in agreement with the relative oxidantpower of each system (FIG. 1 and section 3.1). Increased stress required3× TRIAD instead of 1× to provide complete neuroprotection (FIG. 7).Pyruvate contributed for most of TRIAD protective effect in thisprooxidant system (FIG. 8).

[0117] 3.5 In Vitro Antioxidant Capacity of TRIAD with Peroxide-BasedProoxidant Systems Used with Cultured Neurons

[0118] Antioxidant capacity of TRIAD and of its components wereevaluated in vitro by following oxidation of DPD by the twoperoxide-based prooxidant systems. FIGS. 9 and 10 show that optimalantioxidant concentrations of TRIAD for peroxide systems are smaller invitro than in cell culture situations. Indeed, 0.5× and 1× TRIADrespectively abolished DPD oxidation (FIG. 9) and neuron mortality (FIG.5) induced by oxidative stress in the H₂O₂ system, and the counterpartvalues were 1× (FIG. 7) and 3× (FIG. 10) for the H₂O₂/Fe²⁺ system. Theseobservations suggest that even low levels of oxidative stress couldexert irreversible detrimental effects on cells, requiring higherconcentrations of TRIAD to be prevented.

[0119] Results of DPD oxidation measurement show that there areresemblance and also differences regarding the relative protectionafforded by each component of TRIAD in vitro, compared to cell culturesituations, giving clues on the possible mechanism of action of TRIAD.Differences were obvious with the H₂O₂ system. In cultured neurons,pyruvate mostly (60%) contributed to the neuroprotective action of TRIADagainst H₂O₂-induced injury whereas fatty acids and vitamin E did not bythemselves provide much protection to cells (less than 10% for peroxideconcentration higher than 1 mM) although they increased pyruvate actionsynergistically (section 3.3 and FIG. 6). In contrast, in vitro, fattyacids+vitamin E completely inhibited the oxidation of DPD by H₂O₂ whilepyruvate also provided a substantial although not total antioxidanteffect (FIG. 11). Discrepancy between cultured cells and in vitrosituations could be explained by the presence or absence of a cellmembrane barrier which distinguishes inside and outside protection. Invitro, fatty acids+vitamin E combination and pyruvate can separatelyinhibit DPD oxidation by H₂O₂ because they are all in the samecompartment. In contrast, there are at least two compartments in cellcultures (inside and outside cells). Because pyruvate can be uptaken byneurons, it can protect them from both exterior and interior damagesinduced by an excess of H₂O₂ which is known to diffuse easily throughcell membranes. Fatty acids (including lecithin present in egg yolk) andvitamin E which do not pass easily through membranes during the 30 minof treatment, could not afford important intracellular protection butrather helped pyruvate by providing extracellular defense. Fattyacids+vitamin E combination were only as powerful as pyruvate to inhibitDPD oxidation when iron was added to hydrogen peroxide (FIG. 12). It ispossible that this combination lost part of its antioxidant propertiesbecause egg yolk fatty acids were deteriorated by iron-catalyzedformation of hydroxyl radicals which are known to initiate lipidperoxidation (Gutteridge, 1994; Chan, 1996). Neurons would thus countmore on pyruvate for their protection in the H₂O₂/Fe⁺² than in the H₂O₂system. Unfortunately, the XA/XAO system could not oxidize DPD byitself. Therefore direct comparison between this system and theperoxide-based systems cannot readily be made.

4. Discussion

[0120] This study showed that TRIAD has an antioxidant capacity in vitroand a protective action on cultured P19 neurons exposed to oxidativestress. The results are summarized in Table I below and indicate thatassociation of pyruvate, vitamin E and fatty acids can protect cellsagainst extracellular and intracellular oxidative damages, by differentmechanisms. Since oxidative damage in vivo can be caused byextracellular or intracellular (or both) ROS sources, association of thethree components of TRIAD appears very useful. TABLE I Minimalconcentration of TRIAD (X-fold) for complete antioxidant protection.Mod- Prooxidant system el XA/XAO H₂O₂ H₂O₂/Fe²⁺ Neu- rons

In vitro Not determined*

[0121] Although not shown in this study, the Inventors have demonstratedthat TRIAD protected hearts against oxidative stress generated viaseveral important ROS (.O₂ ⁻, H₂O₂ and .OH), physiologically produced inischemia-reperfusion conditions. Smaller concentrations of TRIAD wasneeded to protect isolated heart from ischemia-reperfusion induceddamages than to protect isolated neurons. There are several explanationsto this difference. First, concentrations of ROS used in this study withneurons were high and likely more important than those encounterednaturally. In addition, ROS produced exogenously have conceivably aneasier access to cells grown as monolayers than to cells tightlyorganized within an organ, and organ being formed of different types ofcells, it likely possesses a larger spectrum of antioxidant defensesthan have monotypic cells in cultures. However, 1× and 3× were veryeffective concentrations of TRIAD in cultured neurons. In peroxide-basedprooxidant systems, concentration dependency of the protective effect ofTRIAD in cultured cells matched that of its antioxidant capacity invitro. However, higher concentrations than those predicted from in vitroanalyses were systematically needed with neurons. These results suggestthat cellular damages can accumulate before TRIAD entirely exerts itsprotective action and/or that cellular metabolism can trigger ROStransformation from one type to a more reactive one.

[0122] Pyruvate was the most important component of TRIAD with culturedneurons, accounting for 60 to 90% of the protective action of TRIAD.Fatty acids and vitamin E by themselves did not provide much protectionbut they increased the protective effect of TRIAD, most often in anadditive but sometimes in a synergistic manner. Pyruvate is consideredas an important scavenger of H₂O₂ and compared to the other agents ofTRIAD, provides important intracellular neuroprotection due to thecapacity of neurons to import pyruvate from extracellular sources. As anexception to the important contribution of pyruvate, antioxidantcapacities of TRIAD in vitro with H₂O₂ prooxidant system was mainlycontributed by fatty acids and vitamin E. Addition of Fe⁺² to H₂O₂diminished the antioxidant power of fatty acids+vitamin E in vitro, aneffect which could be related to a possible peroxidation of egg yolklipids by newly formed hydroxyl radicals.

[0123] The Applicant is aware of the apparent contradiction between theresults obtained with cultured neurons (FIG. 6, where pyruvate is themajor protector) and those obtained with DPD (FIG. 11, where pyruvate isnot the major protector) for H₂O₂ system, as discussed in the precedentparagraph. A possible explanation for this observation is the existenceof at least two compartments in the cell situation (intracellular andextracellular compartments) compared to only one compartment in the testtube assay.

[0124] In vitro, either pyruvate alone or the combination of vitaminE+fatty acids is in concentration sufficient to decrease or prevent DPDoxidation by hydrogen peroxide (FIG. 11). However, damages caused tocells by hydrogen peroxide are intra- as well as extracellular, sincethis ROS can pass through cell membrane. In vivo, pyruvate would provideintracellular protection because it is uptaken by cells, while vitamin Eand fatty acids which do not pass cell membranes provide extracellularprotection only. One could imagine that if cells are permeabilized, thenvitamin E+fatty acids and pyruvate would perhaps provide an overallprotection resembling that seen in vitro. However, the formulation ofTRIAD tested, has the advantage of both extracellular (membrane) andintracellular effects.

[0125] The neuroprotective action of TRIAD is likely related to itsthree components. Pyruvate, able to enter the cell, will enhanceintracellular defense, while vitamin E and fatty acids will improvemembrane functionality, eventually limiting the leakage of cellular Fe²⁺ion (easily generated by reduction of Fe³⁺→Fe²⁺, induced by superoxideanion which is a reductive agent), preventing thus the production ofhydroxyl radical (—OH) via the Fenton and Haber-Weiss reactions,

[0126] Fenton reaction: Fe²⁺→H₂O₂→Fe³⁺+.OH+OH⁻

[0127] Haber-Weiss reaction: Fe³⁺+.O₂ ⁻→Fe²⁺+O₂

[0128] Mechanisms of iron involvement are not fully elucidated, butthere is a growing consensus that oxidative tissue damage is related tonon-heme cellular iron mobilized from cytosolic metal-containing sites:e.g. ferritin stores within cells.

[0129] In this work, the protective effect of TRIAD was studied duringco-exposure of neurons to both prooxidant conditions and TRIAD. In atherapeutic point of view, this antioxidant TRIAD mix could conceivablybe also used to prevent damages caused to tissues by acute or chronicexposure to oxidative stress or to recover from such injuries. In thisaspect, the potential of egg yolk to serve as a source of fatty acids torepair membrane damages and that of pyruvate to serve as fuel for cellscould confer important neurotrophic properties to TRIAD and extentapplication of TRIAD to neurodegenerative diseases. This is particularlyrelevant since oxidative stress is considered as an etiologic or atleast an aggravating factor in several of these diseases. TRIAD thus hasa high therapeutic potential in preventive or reparative strategies.

5. Conclusive Remarks

[0130] This study shows that TRIAD has an antioxidant neuroprotectiveaction on cultured P19 neurons exposed to oxidative stress. Optimalconcentrations vary with the type and prooxidant power of ROS generatingsystems. Pyruvate is a major contributor of antioxidant properties ofTRIAD ex vivo (heart, not shown) and in neuronal cultures, especiallywhen TRIAD is administered just prior induction of an oxidative stressand remains present for short time of treatment (30-40 min for neurons).The contribution of vitamin E and egg yolk fatty acids may appear evenmore important in antioxidant defense when TRIAD is administered forlonger periods (before, during and after oxidative stress).

[0131] This study also yields in the development of an essential conceptwhich comprises two aspects:

[0132] i) combinations of antioxidants having different mechanism ofaction provide higher protection to oxidative stress than any singleantioxidant; and

[0133] ii) synergistic protection is a “latent” property of antioxidantcombinations and does not necessarily manifest itself in all prooxidantconditions.

[0134] Aspect ii) is best illustrated by the results of FIG. 6 whichshowed that while pyruvate and vitamin E+fatty acids each provides halfof the protective effect of TRIAD at low hydrogen peroxideconcentrations, at higher concentrations of the prooxidant, TRIADremained almost as protective even though vitamin E+fatty acids were nolonger active by themselves. Synergistic neuroprotection was thus seenunder more pronounced oxidative stress conditions.

[0135] Finally, although the term “TRIAD” used herein refers to acomposition comprising sodium pyruvate, vitamin E and egg yolk fattyacids, a person skilled in the art will understand that the compositionsof the present invention are not restricted to these sole specificcomponents as explained previously in the first part of the section“DETAILED DESCRIPTION OF THE INVENTION”.

6. REFERENCES

[0136] Throughout this paper, reference is made to a number of articlesof scientific literature which are listed below:

[0137] Anonymous (1985) DPD calorimetric method. Standard methods forthe examination of water and wastewater. New-York, APHA, AWWA, WPCF,16^(th) ed., 306-309.

[0138] Chahine, R., Mateescu, M. A., Roger, S., Yamaguchi, N., DeChamplain, J. and Nadeau, R. (1991) Can. J. Physiol. Pharmacol. 69,1459-1464.

[0139] Chan, P. (1996) Stroke 27, 1124-1129.

[0140] Cini, M., Fariello, R. G., Bianchetti, A. and Moretti, A. (1994)Neurochem. Res. 19, 283-288.

[0141] Desagher, S., Glowinski, J. and Premont J. (1996) J. Neurosci.16, 2553-2562.

[0142] Finley, M. F. A., Kulkarni, N. and Hutter, J. E. (1996) J.Neurosci. 16, 1056-1065.

[0143] Gokhale, M. S., Lin, J. R. and Yager, J. D. (1997) Toxicol. inVitro 11, 753-759.

[0144] Gutteridge, J. M. C. (1994) Annu. N.Y. Acad. Sci. 738, 201-213.

[0145] LeBel, C. P. and Bondy, S. C. (1991) Neurotox. Teratol. 13,341-346.

[0146] Jeannotte, R., Paquin, J., Petit-Turcotte, C. and Day, R. (1997)DNA Cell Biol. 16, 1175-1187.

[0147] Maiese, K. (1998) Clin. Neuropharmacol. 1, 1-17.

[0148] Martin, A. (1994) U.S. Pat. No. 5,926,370.

[0149] Martin, A. (1996) Dermatol. Surg. 22, 156-160.

[0150] McBurney, M. W. (1993) Int. J. Dev. Biol. 37, 135-140.

[0151] Parnas, D. and Linial, M. (1997) Molec. J. Neurosci. 8, 115-130

[0152] Sheridan, J., Kern, E., Martin, A. and Booth, A. (1997) AntiviralRes. 36, 157-166.

[0153] Takemura, G., Onodera, T. and Ashraf, M. (1994) J. Mol. CellCardiol. 26, 41-454.

[0154] Yao, M., Bain, M. Y. G. and Gottlieb, D. I. (1995) J. Neurosci.Res. 41, 792-804.

[0155] Of course, numerous modifications and improvements could be madeto the embodiments that have been disclosed herein above. Thesemodifications and improvements should, therefore, be considered a partof the invention.

1-19. (canceled)
 20. A method for preparing a neuroprotectivecomposition, characterized in that it comprises the steps of: a)providing a therapeutically effective amount of: i) pyruvate, ii) atleast one antioxidant; and iii) at least one lipid; and b) mixingtogether the components i), ii) an iii) of step a) in a physiologicalbuffered saline solution to obtain a pharmaceutically acceptablehomologous suspension.
 21. The method of claim 20, further comprisingcentrifuging the homologous suspension.
 22. The method of claim 20,further comprising filtering the homologous suspension.
 23. Aneuroprotective composition comprising a therapeutically effectiveamount of pyruvate at least one antioxidant and at least one lipid in aphysiological buffered saline solution.